1. Technical Field
The invention is in the fields of drug therapy; more specifically, the invention encompasses a protein for immunosuppression.
2. The Prior Art
The central nervous system (CNS) actively maintains immune privilege (Carson and Sutcliffe, 1999; Fabry et al., 1994), in part by restricting immune cell access (Goldstein and Betz, 1986; Hickey et al., 1991), having limited afferent antigen drainage (Weller et al., 1996; Cserr and Knopf, 1992), locally suppressing immune responsiveness (Irani et al., 1996; Irani et al., 1997), guiding the recruitment and differentiation of effector cell phenotypes (Aloisi et al., 1998; Carson et al., 1999), and possessing weak antigen presenting cells (Carson et al., 1998).
Neurons may directly modulate immune responsiveness. Absence of constitutive neuronal MHC expression may limit anti-neuronal cytotoxic T-cell effector mechanisms (Rall, 1998). Glycosphingolipids known as gangliosides are enriched within neurons, can be shed from the cell surface, are immunosuppressive, and may contribute to immune privilege (Irani et al., 1996; Rall, 1998). Gangliosides suppress the expression of MHC molecules (Massa, 1993), the proliferation of T-cells, and the production of IL-2 (Irani et al., 1996; Irani et al., 1997; Bergelson, 1995; Robb, 1986; Dyatlovitskaya and Bergelson, 1987).
The therapeutic approach of transfecting and transplanting neurons to ameliorate neurological deficits requires a defined, preferably clonal source of differentiated human neurons amenable to efficient transfection and sustained expression of therapeutic genes (Trojanowski et al., 1997; Cook et al., 1994). A therapeutic effect is anticipated should the engrafted cells retain a neuronal phenotype, functionally integrate, and deliver a sustained level of therapeutically relevant protein to the affected region of the brain (Cook et al., 1994). This approach has evolved from trials utilizing neuronal isolates of the embryonic ventral mesencephalon (Kordower et al., 1995; Bjorklund, 1992; Perlow et al., 1979), modified neuronal progenitors (Sabate et al., 1995), neurons (Anton et al., 1994), or fibroblasts (Fisher et al., 1991). Ganglioside shedding and the absence of MHC expression may favor resistance of the neuronal graft to MHC-restricted T-cell attack (Lampson and Siegel, 1988).
Embryonic neurons as grafts are limited by their heterogeneity, expense, scarcity, diminishing viability over time, and refractoriness to standard transfection techniques (Cook et al., 1994; Meichsner et al., 1993). A promising alternative neuron, which is amenable to transfection, is derived from the embryonal carcinoma cell line Ntera2/D1, a putative neuronal progenitor (Cook et al., 1994; Andrews et al., 1984). Ntera2/D1 differentiate in response to treatment with all-trans-retinoic acid into a mixture of cells, including postmitotic cells with a neuronal phenotype (Andrews, 1984; Pleasure et al., 1992; Pleasure and Lee, 1993). Cultures are selectively enriched for Ntera2/D1-derived neurons (designated hNT neurons) by inhibiting the non-neuronal cells with mitotic inhibitors, and by replating hNT neurons on poly-D-lysine plus laminin, which encourages growth of polarized processes. In this manner, cultures comprised of >90% hNT neurons are prepared (Cook et al., 1994).
hNT neurons have identifiable axons and dendrites (Andrews, 1984), retain a plasticity to regenerate and extend neurites after multiple replatings in vitro (Cook et al., 1994), and express neurofilaments characteristic of neuronal development and the adult CNS (Andrews, 1984; Lee and Andrews, 1986). hNT neurons synthesize neurotransmitters, express the catecholamine biosynthetic enzyme tyrosine hydroxylase, and excrete the dopamine metabolite homovanillic acid (Zeller and Strauss, 1995; lacovitti and Stull, 1997). Transplanted hNT neurons are capable of long-term functional integration (Kleppner et al., 1995), are nontumorigenic (Trojanowski et al., 1997), and can correct behavioral deficits in the lesioned rodent (Borlongan et al., 1998).
Although a therapeutic potential of hNT neuronal grafts has been implied, a paucity of data exists regarding its MHC and immunological features. Retinoic acid-induced differentiation of Ntera2/D1 causes the produced hNT neurons to express MHC class I and β-2 microglobulin molecules (Segars et al., 1993), but whether hNT neurons express a discernable MHC phenotype that can activate allogeneic immunocytes has not been determined. An increase in the expression of gangliosides (e.g., GD3 and GT3) and the glycolipid sialyltransferases that contribute to their synthesis occurs during the differentiation of some embryonal carcinoma cells (Chen et al., 1989; Osania et al., 1997). Whether hNT neurons can modulate immune responses and shed gangliosides at immunosuppressive levels have not been determined. Some CNS neoplasms (e.g., gliomas) express immunosuppressive levels of transforming growth factor-β (TGF-β) (Weller and Fontana, 1995). TGF-β inhibits T-cell proliferation by suppressing IL-2-mediated proliferative signals (Ahuja et al., 1993). Retinoic acid treatment increases TGF-β expression during murine embryogenesis (Mahmood et al., 1995), and during embryonal carcinoma cell differentiation (Rizzino et al., 1983), but whether hNT neurons express immunosuppressive levels of TGF-β has not been determined.